Fluorescent chemosensors are dye molecules whose fluorescence excitation/emission changes in response to the surrounding medium or through specific molecular recognition events. See Molecular Fluorescence: Principles and Applications. , Valeur, B.; Wiley-VCH: New York (2001). Due to their simplicity and high sensitivity, fluorescent sensors have been widely utilized as popular tools for chemical, biological, and medical applications. See Molecular Fluorescence: Principles and Applications. , Valeur, B.; Wiley-VCH: New York (2001); Principles of Fluorescence Spectroscopy, 2nd ed., Lakowicz, J. R., Kluwer Academic/Plenum: New York (1999); and Fluorescent Chemosensors for Ion and Molecule Recognition, Czarnik, A. W., American Chemical Society: Washington, DC (1993). The most general strategy for fluorescent sensor design is to combine fluorescent dye molecules with designed receptors for specific analytes, in hopes that the recognition event between receptor and analyte will lead to a fluorescence property change of the dye moiety. Although many fluorescent sensors have been successfully developed through this approach, each individual development requires a major effort in both the design and synthesis of the sensors. Also, the sensor's scope of application is limited to the selected specific analytes that the sensor was rationally designed for; these are so-called Analyte Directed Sensors. See Srinivasan, N., et al., Curr. Opin. Chem. Biol., 8: 305 (2004); Rurack, K., et al., Chem. Soc. Rev., 31, 116 (2002); Valeur, B., et al., Coord. Chem. Rev., 205: 3 (2000); Martinez-Manez, R., et al., Chem. Rev., 103: 4419 (2003); and de Silva, A. P., et al., Chem. Rev., 97: 1515 (1997). Combinatorial dye library synthesis offers one of the most promising alternatives, once an efficient synthetic route can be developed for a diverse set of dyes. Sensors developed using this approach are called Diversity Directed Sensors. Combinatorial chemistry is now widely being used in the chemical biology and medicinal/pharmaceutical field for the discovery of biologically active molecules or drug candidates, yet the application of this method to fluorescent dyes is only in its infancy. See Li, Q., et al., Angew. Chem. Int. Edit., 43: 6331 (2004); Gao, J., et al., J. Am. Chem. Soc., 126: 12748 (2004); Rosania, G. R., et al., J. Am. Chem. Soc., 125: 1130 (2003); and Zhu, Q., et al., Tetrahedron Lett., 43: 5083 (2002).
Nucleotide anion detection has long intrigued researchers and witnessed continuous growth. See Li, C., et al., Angew. Chem. Int. Edit., 44: 6371 (2005); Descalzo, A. B., et al., J. Mater. Chem., 15: 2721 (2005); Mizukami, S., et al., J. Am. Chem. Soc., 124: 3920 (2002); Ojida, A., et al., Tetrahedron Lett., 43: 6193 (2002); Sancenon, F., et al., Helv. Chim. Acta, 85: 1505 (2002); Thanh, N., et al., Anal Lett., 35: 2499 (2002); Turkewitsch, P., et al., J. Photochem. Photobiol., 117: 199 (1998); Kim, S. K., et al., Tetrahedron Lett., 46: 6617 (2005); Kwon, J. Y., et al., J. Am. Chem. Soc., 126: 8892 (2004); McCleskey, S. C., et al., J. Am. Chem. Soc., 125: 1114 (2003); and Amemiya, S., et al., Chem. Commun., 1027 (1997). Although GTP plays an important role in biological processes, very little work has been done on the development of fluorescent sensors for it. See Kim, S. K., et al., Tetrahedron Lett., 46: 6617 (2005); Kwon, J. Y., et al., J. Am. Chem. Soc., 126: 8892 (2004); McCleskey, S. C., et al., J. Am. Chem. Soc., 125: 1114 (2003); Amemiya, S., et al., Chem. Commun., 1027 (1997); Burma, D. P., J. Sci. Ind. Res., 47: 65 (1988); and Pogson, C. I., Am. J. Clin. Nutr., 27: 380 (1974). Thus far, the best reported GTP sensor, which was designed rationally, showed around 90% quenching response at around mM concentration of GTP, and most of the known GTP sensors compete with ATP to some extent. See Kwon, J. Y., et al., J. Am. Chem. Soc., 126: 8892 (2004). Currently, no turn-on fluorescent sensors for GTP have been reported yet.
Heparin is a naturally occurring polysaccharide which has been used as a major anticoagulant to prevent and treat thrombotic diseases since early 20th century. See Capila, I., et al., Angew. Chem., Int. Ed., 41: 391 (2002); Whitelock, J. M., et al., Chem. Rev., 105: 2745 (2005); and Rabenstein, D. L., Nat. Prod. Rep., 19: 312 (2002). It is considered second only to insulin in the terms of being a very successful natural therapeutic agent. See Rabenstein, D. L., Nat. Prod. Rep., 19: 312 (2002). Despite its long history and wide use, closely monitoring and control of the Heparin blood levels during the application of unfractionated heparin (UFH) and Low Molecular Weight Heparin (LMWH) is of crucial importance due to the risk of adverse effects such as hemorrhages and heparin-induced thrombocytopenia (HIT) resulting from overdoses. See Warkentin, T. E., et al., New England Journal of Medicine, 332: 1330-1335 (1995); Hoppensteadt, D., et al., Hematology-Oncology Clinics of North America, 17: 313 (2003); and Pineo, G. F., et al., Medical Clinics of North America, 82: 587 (1998). Various assays have been established to monitor the heparin concentration, including the most commonly used assays: activated partial thromboplastin time (aPTT), anti-Xa, and activated clotting time (ACT) assays. See Simko, R. J., et al., Annals of Pharmacotherapy, 29: 1015-1021 (1995); Murray, D. J., et al., Journal of Cardiothoracic and Vascular Anesthesia, 11: 24-28 (1997); and Marci, C. D., et al., American Journal of Clinical Pathology, 99:546-550 (1993). Although the evolution of methods for monitoring heparin has been improving through the decades, which method is the ideal remains controversial. See Kitchen, S., British Journal of Haematology, 111: 397-406 (2000); and Francis, J. L., et al., Pharmacotherapy, 24: 108-119 (2004).
Fluorescent chemosensors have witnessed a continuous progress together with the development of supermolecular chemistry and molecular recognition throughout the decades. See Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic/Plenum: New York (1999); and Valeur, B., Molecular Fluorescence: Principles and Applications, Wiley-VCH: Weinheim; New York (2002). Various chemosensors have been developed and successfully utilized in diverse chemical, biological and medical applications. The properties of fluorescence, such as simplicity and high sensitivity, are highly appreciated. An important field for chemosensors is the targeting of bio-relevant analytes. The development of chemosensors for heparin assay was marked by the pioneering work of Anslyn's Group. See Zhong, Z. L., et al., J. Am. Chem. Soc., 124: 9014 (2002); and Wright, A. T., et al., Angewandte Chemie-International Edition, 44: 5679-5682 (2005). A tripodal boronic acid based small molecule with intramolecular boron-nitrogen interaction was designed and synthesized for heparin assay and was demonstrated as a fluorescence quenching sensor, which for the first time raised the question of fluorescent sensing heparin. Along these lines, a peptide based sensor was developed based on a heparin-specific peptide sequence AG73. See Sauceda, J. C., et al., Chembiochem, 8: 391-394 (2007). Chloride anion quenched fluorescence was regenerated when heparin introduced. However, these sensors were carefully designed for the specific purpose and, as previously noted, Analyte Directed Sensors. Combinatorial dye library synthesis offers one of the most promising alternatives as Diversity Directed Sensors, once an efficient synthetic route can be developed for a diverse set of dyes.
The present invention is directed to overcoming these and other deficiencies in the art.